It is well recognized that the "spectrin-actin junctions" are functionally essential for the maintenance of erythrocyte membrane stability and mechanical properties. The prevailing paradigm is that the "spectrin-actin junctions" are linked to the plasma membrane via an interaction between protein 4.1 and glycophorin C. Based on our recent findings, we propose an alternate model for the vertical linkage of "spectrin-actin junctions" to the plasma membrane. Our current model predicts that dematin, in conjunction with adducin, assembles a novel cytoskeletal complex at the tail ends of spectrin polypeptides that links the [unreadable]spectrin-actin junctions[unreadable] to the plasma membrane via a transmembrane protein. In this competitive renewal, the following experimental approaches will be used to validate our hypothesis: (1) Combined deletion of mouse dematin and adducin results in severe anemia with highly fragile erythrocytes. To elucidate the mechanism of weakened spectrin-actin linkage to the plasma membrane, we will investigate the status of actin protofilaments, rescue membrane instability defect, examine the effect of double mutation on known vertical interactions, and test direct binding of dematin with adducin and its regulation by phosphorylation and oligomerization. (2) Dematin binds to a protease-sensitive site on the erythrocyte plasma membrane. To identify this protein, we will perform surface labeling of cells, immunoprecipitations, protein pull-down assays, and proteomic analysis of erythrocyte membrane vesicles. A similar analysis will be performed for adducin. These experiments are likley to unveil new transmembrane proteins that would tether the junctional complex to the plasma membrane. (3) Following the strategy outlined in Aim 2, we detected a 50 kDa erythrocyte surface protein in the immunoprecipitates of dematin. Further analysis indicates that this protein is glucose transporter 1 (GLUT-1), a major transmembrane receptor of the erythrocyte membrane. We propose to characterize the biochemical nature of human dematin and adducin interactions with GLUT-1, identify the interacting domains, and demonstrate the conservation of the new bridge in non-erythroid cells. Since dematin, adducin, and GLUT-1 are widely expressed, including the brain, the proposed studies are potentially significant for understanding the molecular basis of blood-brain barrier glucose transport in normal and pathological settings such as GLUT-1 deficiency syndrome.